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Genes to Cells (2007) 12, 1357-1370. doi:10.1111/j.1365-2443.2007.01141.x
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Rapamycin sensitivity of the Schizosaccharomyces pombe tor2 mutant and organization of two highly phosphorylated TOR complexes by specific and common subunits

 Takeshi Hayashi1,3,a, Mitsuko Hatanaka1,3,a, Koji Nagao1,*,a, Yukinobu Nakaseko3, Junko Kanoh3, Aya Kokubu1, Masahiro Ebe2,3 and Mitsuhiro Yanagida1,2,3,*

1 The G0 Cell Unit, Okinawa Institute of Science and Technology Promotion Corporation, Suzaki 12-22, Uruma, Okinawa 904-2234, Japan
2 CREST Research Program, Japan Science and Technology Corporation, and
3 The Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan.


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Nutrients are essential for cell growth and division. Screening of Schizosaccharomyces pombe temperature-sensitive strains led to the isolation of a nutrient-insensitive mutant, tor2-287. This mutant produces a nitrogen starvation-induced arrest phenotype in rich media, fails to recover from the arrest, and is hypersensitive to rapamycin. The L2048S substitution mutation in the catalytic domain in close proximity to the adenine base of ATP is unique as it is the sole known genetic cause of rapamycin hypersensitivity. Localization of Tor2 was speckled in the vegetative cytoplasm, and both speckled and membranous in the arrested cell cytoplasm. Using mass spectroscopic analysis, we identified six subunits (Tco89, Bit61, Toc1, Tel2, Tti1 and Cka1) that, in addition to the six previously identified subunits (Tor1, Tor2, Mip1/Raptor, Ste20/Rictor, Sin1/Avo1 and Wat1/Lst8), comprise the TOR complexes (TORCs). All of the subunits so far examined are multiply phosphorylated. Tel2 bound to Tti1 interacts with various phosphatidyl inositol kinase (PIK)-related kinases including Tra1, Tra2 and Rad3, as well as Tor1 and Tor2. Schizosaccharomyces pombe TORCs should thus be functionally redundant and might be broadly regulated through different subunits that are either common or specific to the two TORCs, or even common to various PIK-related kinases. Functional redundancy of the TORCs may explain the rapamycin hypersensitivity of tor2-287.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The target of rapamycin (Tor) kinases have an essential role in cell growth by regulating the cellular response to changes in available nutrients (Jacinto & Hall 2003; Fingar & Blenis 2004; Wullschleger et al. 2006). The two evolutionarily conserved TOR complexes, TORC1 and TORC2, are present in eukaryotes. Although only one core subunit, mTOR, exists in higher eukaryotes (Loewith et al. 2002; Kim et al. 2003; Jacinto et al. 2004; Sarbassov et al. 2004), the budding yeast Saccharomyces cerevisiae has two catalytic subunit genes (Heitman et al. 1991; Kunz et al. 1993; Helliwell et al. 1994). The fission yeast Schizosaccharomyces pombe has also two catalytic subunit genes, tor1+ and tor2+ (Kawai et al. 2001; Weisman & Choder 2001; Alvarez & Moreno 2006; Uritani et al. 2006; Matsuo et al. 2007; Weisman et al. 2007). Schizosaccharomyces pombe is an excellent organism to investigate the TOR systems as this organism contains homologues of Rheb and TSC (Mach et al. 2000; Yang et al. 2001; Matsumoto et al. 2002), which regulate the Tor kinase activities in higher eukaryotes. It is uncertain whether S. cerevisiae has typical Rheb and TSC homologues (Aspuria et al. 2007).

To investigate the regulation of non-dividing cells under nitrogen-starved conditions (Su et al. 1996; Shimanuki et al. 2007), we searched for temperature-sensitive (ts) mutants with phenotypes similar to those of nitrogen-starved arrested cells in complete medium at a restrictive temperature (36 °C). One such ts mutant strain was isolated, and gene cloning indicated that the mutant gene was tor2. The tor2 mutant was hypersensitive to rapamycin at the permissive temperature (26 °C) and produced a similar phenotype at 36 °C. We characterized this mutant, which is unique in regard to its rapamycin hypersensitivity. To understand the role of Tor2 in dividing and non-dividing cells, information about the molecular organization of TORCs in this organism is essential. Interpretation of the phenotypic results definitively requires such information. For this end, mass spectroscopic analysis was done, and we identified six new proteins that interacted with the S. pombe TORCs. Our results strongly suggested that two TORCs in S. pombe are functionally overlapping as four of the eight subunits are common. The S. pombe TORCs per se are an excellent model for understanding the regulation of growth by TORCs in higher eukaryotes.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
The arrest phenotype displayed by an Schizosaccharomyces pombe ts mutant in complete medium

To isolate mutants that produce a phenotype similar to that produced under nitrogen-starvation arrest (round shape and 1C DNA; Su et al. 1996) in complete medium, we collected 1015 ts mutant strains of S. pombe (Hayashi et al. 2004; Yuasa et al. 2004). Eighty-five strains with the round shape at 36 °C were first selected, and their DNA contents were measured by FACScan (Becton Dickinson, Franklin Lakes, NJ) after 6 h in complete medium at 36 °C. One mutant (287) that produced a 1C DNA peak was investigated further: micrographs of cells and the DNA content are shown in Fig. 1A,B. After shifting to 36 °C in rich YPD medium, the cell number increase completely stopped after 1–2 cell divisions, although cell viability remained nearly 100% after 24 h (data not shown), suggesting that the cell division arrest is not lethal to this strain in this time span. Basically the same results were obtained in two different culture media, synthetic EMM2 and rich YPD.


Figure 1
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  		Figure 1  Phenotypes of S. pombe ts tor2-287 and the mutation site. (A) Cellular DNA content of tor2-287 cultured at 26 °C analyzed by the FACScan was mostly 2C (bottom panel), but when cultured at 36 °C for 6 h (top panel) in the rich YPD medium, ~20%–30% of cells contained 1C DNA. (B) Mutant tor2-287 cells grown at 26 °C were normal rod-shaped (left), while pear-shaped cells were produced at 36 °C for 6 h in the complete medium EMM2. DNA was stained by DAPI. Bar = 10 µm. (C) and (D) In the nitrogen-deficient medium (EMM2–N), tor2-287 cells normally responded to nitrogen starvation at 26 °C as the shape of cells was round (C, left panel) and the DNA content was mostly 1C (D, 0 h in the left panel). When cells were replenished with the nitrogen source (+N) and cultured at 26 °C or 36 °C, resulting cells were either rod-shaped at 26 °C or pear-shaped at 36 °C (C, right panel). Bar = 10 µm. The DNA content was measured after the nitrogen source replenishment. At 26 °C, DNA content increased at 12 h, but at 36 °C replication was impaired in a considerable fraction of cell populations (D). (E) The mutation site of tor2-287 determined by nucleotide sequencing of the mutant tor2 gene was the Leu residue at amino acid 2048, which was altered to Ser. The mutation site resides in the catalytic domain of TOR kinase.

 
Genetic analysis indicated that the Ts– and arrest phenotypes were co-segregated after backcrossing with the wild-type, indicating that the ts mutation caused the arrest phenotypes observed. At 26 °C in complete media, the strain 287 cells had normal rod-shaped phenotypes with mostly 2C DNA. The cell number increased with the normal generation time (data not shown). These phenotypes suggested that the mutant cells were unable to properly utilize the nutrient at 36 °C.

Strain 287 fails to enter the proliferation phase

To examine whether strain 287 cells can normally exit from nitrogen-starved arrest, strain 287 was first cultured in nitrogen-deficient medium, EMM2–N at 26 °C for 24 h. The resulting arrested cells were round (Fig. 1C left panel) and contained 1C DNA (Fig 1D, 0 h). They were then shifted to complete EMM2 at 26 °C or 36 °C. After 12 h at 36 °C, however, the mutant cells did not grow, whereas the elongated rod-shaped vegetative cells appeared after 12 h at 26 °C (Fig. 1C, right). The mutant cells did not seem to enter the proliferation stage at 36 °C. Only partial DNA replication occurred at 36 °C, whereas full DNA replication occurred at 26 °C (Fig. 1D). The cell viability of the strain 287 cells, however, remained nearly 100%, even 24 h after the shift to EMM2 at 36 °C (data not shown). The block of growth initiation at 36 °C in complete medium was thus not a lethal event for the G0 arrested mutant, and proliferation could be restored if the cells were transferred back to 26 °C. Thus, the mutant nitrogen-starved cells did not properly respond to nitrogen replenishment at 36 °C.

Strain 287 is a tor2 mutant

To identify the mutant gene, plasmids that suppressed the ts phenotype of 287 were isolated by transformation using an S. pombe genomic library. Plasmid DNAs were recovered from the Ts+ transformants and sequenced. The subcloned plasmids contained only the tor2+ gene, one of the two Tor-like genes present in the genome of S. pombe. To verify that the plasmid carrying the tor2+ gene was not a high copy suppressor, tetrads were dissected for the cross between strain 287 and {Delta}rad3 (the rad3+ gene is ~5 kb apart from tor2+), and no recombinant (PD : NPD : TT = 35 : 0 : 0) was obtained for the Ts– and Ura+ phenotypes from the cross between h+ rad3:ura4+ ura4-D18 and h– 287 ura4-D18. The 287 locus was thus concluded to be tor2, and we designated strain 287 as tor2-287.

To identify the mutation site, the mutant gene of tor2-287 was isolated by PCR amplification and the sequence was determined. A single nucleotide change was identified at the 2048th codon, which caused a substitution from the conserved hydrophobic Leu residue to hydrophilic Ser in the catalytic domain (Fig. 1E). In the three-dimensional structure of the catalytic kinase domain, the mutated residue is located close to the ATP-binding region (Walker et al. 1999; see Discussion).

To confirm that the L2048S substitution brought about the phenotype observed in tor2-287, the L2048S mutation was introduced into the wild-type using a chromosome integration vector carrying the kanamycin resistance marker (Nagao et al. 2004). Resulting KanR haploid transformants designated tor2-L2048S[KanR] produced the identical ts phenotypes with a round cell shape and 1C DNA at 36 °C (data not shown). We therefore concluded that the single amino acid substitution L2048S caused the phenotypes described for tor2-287. In the following study, the two strains, tor2-287 and tor2-L2048S[KanR], were used.

Hypersensitivity of tor2-287 and tor2-L2048S[KanR] mutant strains to rapamycin

Both the tor2-287 and tor2-L2048S[KanR] strains were hypersensitive to rapamycin at 26 °C (Fig. 2A,B). They were unable to form colonies in a low drug concentration (0.01 µg/mL) at 26 °C. The wild-type strain 972 was not affected at either 26 °C or 30 °C in a wide range of drug concentrations (Fig. 2B). Dimethylsulfoxide (DMSO) was used as the solvent to dissolve rapamycin and as the control. At the semi-permissive temperature (30 °C), even lower concentrations (1–5 ng/mL) of rapamycin were still inhibitory to colony formation (Fig. 2B).


Figure 2
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  		Figure 2  Mutants tor2-287 and tor2-L2048S[KanR] are rapamycin hypersensitive. (A) Serial dilutions of wild-type strain 972, ts mutants tor2-287 and tor2-L2048S[KanR] cells were spotted on the YPD plate containing 0.2 µg/mL rapamycin or the control solvent DMSO, and cultured at 26 °C or 36 °C. Both mutants failed to produce colonies at 26 °C in the presence of rapamycin. (B) Serial dilutions of wild-type 972 and mutant tor2-287 cells were spotted on the plates containing a series of rapamycin concentrations (0.001–0.1 µg/mL) at 26 °C and 30 °C. Tor2-287 cells failed to grow in the presence of rather low concentrations (5–10 ng/mL) of rapamycin at 26 °C. (C) The wild-type, tor2-287 and tor2-L2048S[KanR] strains were cultured in the presence of rapamycin (0.2 µg/mL) at 26 °C for 6 h, and their cell shape (Normaski and DAPI) and DNA contents (FACS) were examined. The control culture contained the solvent DMSO instead of rapamycin. Bar = 10 µm.

 
We then examined the cellular phenotypes of tor2-287 and tor2-L2048S[KanR] in the presence of rapamycin (0.2 µg/mL) at 26 °C for 6 h, and found that a significant fraction (~30%) of cells contained the 1C DNA peak, and many cells were pear-shaped (Fig. 2C), suggesting that rapamycin caused a phenotype at 26 °C similar to that produced at 36 °C in the absence of drug. The wild-type cells showed certain cell shape changes, but the increase in cell number was normal and the 1C DNA peak was not observed with the drug concentration examined. Recently, three groups (Alvarez & Moreno 2006; Uritani et al. 2006; Matsuo et al. 2007) reported the isolation of S. pombe tor2 ts mutants with cellular phenotypes similar to these mutants at the restrictive temperature, but none of the mutants were hypersensitive to rapamycin and the mutations did not have the L2048S substitution.

Detection of FLAG-tagged Tor2 in extracts of growing and G0-arrested cells

To identify Tor2 protein in cell extracts, the amino terminus of the tor2+ gene was tagged with green fluorescent protein (GFP), and the resulting construct was chromosomally integrated to replace the native gene by homologous recombination using the integration vector (Nagao et al. 2004). The resulting integrant strain GFP-Tor2, which contained the native promoter, was verified by nucleotide sequencing. Because the integrant strain grew normally, the tagged tor2+ gene was functional. A similar strategy was used to construct a strain carrying the chromosomally integrated GFP-tagged Tor1 gene under the native promoter, but the resulting integrant seemed not to be functional (sterile, like the deletion mutant), so we did not use the GFP-Tor1 integrant strain in subsequent experiments. FLAG-tagged Tor1, however, was functional, like FLAG-tagged Tor2, so that the gene products produced by the FLAG-tagged Tor1 and Tor2 chromosomally integrated and expressed under the native promoters were immunochemically examined.

FLAG-tagged Tor1 and Tor2 proteins detected in extracts of S. pombe cells are shown in Fig. 3A. Exponentially growing cells in the vegetative phase (Veg) or in arrested cells under nitrogen-starvation (G0) were examined by immunoblot using antibodies against FLAG. {alpha}-Tubulin was used as the loading control. The FLAG-tagged Tor1 and Tor2 protein bands were observed at the positions expected from their molecular weights. Both Tor1 and Tor2 protein were present in the arrested G0 cells, and their levels were similar to those in the vegetative condition.


Figure 3
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  		Figure 3  The level and localization of Tor proteins in vegetative and nitrogen-starved cells. (A) Both tor1+ and tor2+ genes were tagged with FLAG and chromosomally integrated under the native promoter. Immunoblot patterns of extracts of the integrant strains using antibodies against FLAG. Cells were cultured vegetatively (Veg) or under nitrogen-starvation (G0). {alpha}-Tubulin detected by anti-{alpha}-tubulin antibodies was used as the loading control. (B) The tor2+ gene was tagged with GFP and chromosomally integrated under the native promoter. Intracellular localization of GFP-Tor2 was observed in the vegetative cells (Veg) and under nitrogen-starvation (G0). No-tag cells were observed as negative control. DNA was stained by DAPI. The merged images for GFP-Tor2 (green) and DNA (purple) are also shown. Bar = 10 µm.

 
Intracellular localization of Tor2 in vegetative and G0 arrested cells

As the chromosomally integrated GFP-Tor2 gene expressed under the native promoter was functional, its intracellular localization was examined in the vegetative (Veg) and nitrogen-induced arrested (G0) cells. The DNA was stained with DAPI. GFP-Tor2 displayed cytoplasmic but non-homogenous signals in the vegetative cells (Fig. 3B), suggesting that GFP-Tor2 is not soluble in the cytoplasm. Certain signals appeared speckled, but no known organella in the published literature could be identified in the images. In the nitrogen-starved non-dividing cells, GFP-Tor2 signals were also cytoplasmic and speckled, but in this case membranous signals were more prominent, particularly in the peri-nuclear regions.

Ten proteins interacted with Tor2 and/or Tor1

To understand the role of Tor2 and particularly the rapamycin hypersensitivity of tor2-287, we considered that information about the molecular organization of TORCs is essential, as only Tor1 has been shown to be sensitive to rapamycin in S. pombe (Weisman et al. 2005). Interpretation of the mutant phenotypes may be only possible through information about the subunit composition of TORC2 that contains Tor1. For this end, mass spectroscopic analysis was done for both TORC1 and TORC2. We could determine molecular organization of the two complexes that contained six new proteins. To examine the proteins that are associated with Tor2 and to compare them with those bound to Tor1, extracts of the strains carrying the chromosomally integrated FLAG-Tor2 or FLAG-Tor1 under the native promoter described above were immunoprecipitated using anti-FLAG antibodies. Coomassie Brilliant Blue-stained gel patterns of the precipitated protein are shown in Fig 4A. We used the two control strains that were integrated with the S. pombe histone acetylase 1 (Hat1)-FLAG gene or the Mis18-FLAG, both under the native promoter. Hat1 and Mis18, respectively, are a histone acetylase and centromere protein having no functional relationship to the Tor proteins so that the Tor-specific co-precipitated proteins could be selected by comparison in the proteomic analysis. Extracts of these strains were made simultaneously, and precipitated using anti-FLAG antibodies by the same procedures. Resulting bead-bound proteins were washed and eluted by the FLAG peptide, and run in SDS-PAGE. The 17 or 19 gel slices for each lane were subsequently analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS; ThermoFisher LTQ linear ion trap mass spectrometer, San Jose, CA) after tryptic digestion, as previously described (Obuse et al. 2004).


Figure 4
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  		Figure 4  Proteins co-precipitated with FLAG-tagged Tor1, Tor2 or Tel2. Schizosaccharomyces pombe strains chromosomally integrated with the FLAG-Tor1, FLAG-Tor2, or Tel2-FLAG gene under the native promoter were constructed, and their cell extracts were prepared from asynchronously-growing cells cultured in YPD medium at 33 °C. (A) Proteins bound to FLAG-tagged Tor1 (right) and Tor2 (left) were run in SDS-PAGE, and stained with Coomassie Brilliant Blue. For the control strains, S. pombe strains chromosomally integrated with the Hat1-FLAG or Mis18-FLAG gene were used. Proteins that co-precipitated specifically with either or both FLAG-Tor1 and FLAG-Tor2 were identified (see Experimental procedures). A vertical line with protein names indicates the position in the gel pieces in which they were most often detected. (B) The sequence coverage and emPAI value of the proteins shown in A. (C) Tel2-myc was co-immunoprecipitated with FLAG-Tor2 (left panel) and FLAG-Tor1 (right panel). Tel2-myc, FLAG-tor2 tel2-myc and FLAG-tor1 tel2-myc strains were cultured in YPD medium at 33 °C. Whole cell extracts were prepared and FLAG-Tor proteins were immunoprecipitated with anti-FLAG antibodies. (D) (Left panel) Proteins bound to Tel2-FLAG were separated by SDS-PAGE with control immunoprecipitation and stained with Coomassie Brilliant Blue. Proteins specifically associated with Tel2-FLAG were identified by LC-MS/MS. A vertical line with protein names indicates the position in the gel pieces in which they were most often detected. (Right panel) The sequence coverage and emPAI value of the identified proteins.

 
FLAG-Tor1 precipitated seven proteins; Ste20, Sin1, SPCC777.08c, Wat1/Pop3, SPCC622.13c, Tel2 and Orb5, in addition to Tor1 (Fig. 4B). FLAG-Tor2 also precipitated seven proteins; SPBP18G5.03, SPCC162.12, Mip1, Pop3/Wat1, SPCC622.13c, Tel2 and Orb5, in addition to Tor2. Four proteins (Wat1, SPCC622.13c, Tel2, Orb5) were commonly present in both precipitates so a total of 10 different proteins were identified as Tor1, Tor2-interacting proteins. The names of the proteins are listed with the values of sequence coverage (percentage of the protein sequence covered by identified peptides), and the exponentially modified protein abundance index (emPAI) values defined by Ishihama et al. (2005). The emPAI values were calculated from 10PAI-1 (PAI = observed peptide number/observable peptide number). Mass spectroscopic results suggested that Tor1 and Tor2 formed independent complexes, as no specific Tor1 or Tor2 peptide was present in the FLAG-Tor2 or FLAG-Tor1 precipitates, respectively, while they bind to four common proteins.

Common components of TORC1 and TORC2

Previous studies demonstrated that S. pombe Tor1 is associated with Ste20, Sin1 and Wat1, whereas Tor2 is associated with Mip1 and Wat1 (Alvarez & Moreno 2006; Aspuria et al. 2007; Matsuo et al. 2007). In addition to Mip1 (similar to mammalian Raptor and budding yeast Kog1), two proteins, SPBP18G5.03 and SPCC162.12, were specifically associated with Tor2. SPBP18G5.03 was designated as Toc1 (Tor complex). We did not find a clear homologue of Toc1 in the NCBI nr-aa database, even though the homologue exists in the S. japonicus genome ( Schizosaccharomyces japonicus Sequencing Project); the sequence comparison is shown in Supplementary Figure S1. The other protein, SPCC162.12, is similar to TCO89 (89 kDa subunit of Tor complex one), a component of S. cerevisiae TORC1 (Reinke et al. 2004), and is hereafter designated Tco89. Orthologues of Tco89 were found in fungi and these proteins share two conserved domains (Supplementary Figure S2).

Ste20 was similar to mammalian Rictor and budding yeast Avo3, and Sin1 was similar to budding yeast Avo1. An additional protein, SPCC777.08c, was specifically bound to FLAG-Tor1. It was similar to S. cerevisiae BIT61, the component of TORC2, and also to Aspergillus nidulans septation controlling gene HbrB, and so it was designated Bit61 (Supplementary Figure S3). Human oncogene PRR5/Protor-1 also belongs to the BIT61/HbrB family. Schizosaccharomyces pombe has another BIT61/HbrB-like protein, but it was not detected in either the FLAG-Tor1 or FLAG-Tor2 precipitates.

In addition to the previously reported Wat1 (similar to the budding yeast Lst8), three proteins, SPCC622.13c (conserved eukaryotic protein similar to human KIAA0406 and budding yeast YKL033W), Tel2/SPAC458.03 (Shikata et al. 2007) and Cka1 (casein kinase II /Orb5; Snell & Nurse 1994) were associated with both TORC1 and TORC2. Tel2 and Cka1/Orb5 are essential for viability. To our knowledge, these three conserved proteins are novel interacting components of TORC1 and TORC2.

Tel2 was co-precipitated with Tor1 and Tor2

Fission yeast Tel2 is implicated in the regulation of Rad3, a phosphatidyl inositol kinase (PIK)-related damage checkpoint kinase, through the phosphorylation of Mrc1 (similar to human Claspin, a mediator of DNA replication checkpoint kinases) and Chk2/Cds1 checkpoint kinase (Shikata et al. 2007). The tel2+ gene is essential for viability, whereas the rad3+ gene is not essential, indicating that Tel2 has roles other than as a DNA replication checkpoint. Therefore, to confirm that Tel2 was really associated with the TORCs, immunoprecipitation was performed using the strains constructed by the chromosomal integration of Myc-tagged Tel2 (Shikata et al. 2007) and FLAG-tagged Tor2 under the native promoter. Immunoprecipitation was performed using anti-FLAG antibodies, and immunoblotting was performed using antibodies against Myc (Tel2) and FLAG (Tor2). FLAG-Tor2 co-precipitated with Tel2-Myc (Fig. 4C, left panel). Similarly, FLAG-Tor1 was precipitated with Tel2-Myc (the level of Tel2 precipitation by Tor1 was somewhat low).

LC-MS/MS identification of Tel2-interacting proteins

To examine the proteins bound to Tel2, LC-MS/MS analysis was applied to the Tel2-interacting proteins. FLAG-tagged Tel2 was chromosomally integrated and expressed under the native promoter, and the resulting strain was employed for immunoprecipitation using anti-FLAG antibodies. The SDS-PAGE gel pattern and the list of co-precipitated proteins are presented in the left and right panels of Fig. 4D, respectively. Two relatively abundant proteins, SPCC622.13c and SPBC1604.17c, contained no known protein motif. SPCC622.13c was present in both Tor1 and Tor2 interacting proteins. As the emPAI value of SPCC622.13c was high, the same as that of Tel2, we designated SPCC622.13c as Tti1 (Tel two interacting protein). The emPAI value of SPBC1604.17c was comparable to that of Tor2, 50-fold smaller that that of Tti1, and designated as Tti2. Tti2 was a conserved protein, like Tel2 and Tti1, in budding yeast and humans (Supplementary Figure S4).

In addition to the two proteins described above, four other proteins: SPBP16F5.03c, Wat1/Pop3, Tor2 and SPAC1F5.11c, had a range of emPAI values between 1.7 and 4.4 (the sequence coverage, 25%–44%). SPBP16F5.03c and SPAC1F5.11c are very large PIK-related kinases (PIKKs; ~3500 aa), uncharacterized in S. pombe but similar to budding yeast Tra1 and mammalian TRRAP (Grant et al. 1998; McMahon et al. 1998; Vassilev et al. 1998). We refer, hereafter, to SPBP16F5.03c and SPAC1F5.11c as Tra1 and Tra2 in S. pombe, respectively. Three other PIKKs, Tor1, Rad3 (similar to ATR) and Tel1 (similar to ATM), were also present in the immunoprecipitates, although their emPAI values were smaller than those of the first four proteins. The S. pombe genome has a total of six genes encoding PIKK that contained the FAT and FATC domains (Bosotti et al. 2000), and all of them were present in the Tel2-coprecipitated proteins. Proteins that specifically interact with Tor1 or Tor2, such as Mip1 and Ste20, were not detected in Tel2-FLAG immunoprecipitate. Tel2 might be the regulator of PIKKs rather than the specific binding protein of TORCs. Although the level of Tel1 was at the limit of detection, the level of Tel1 present in cell extracts might be rather low.

Phosphorylation of Tor1, Tor2 and associated proteins

To determine whether Tor1, Tor2 and their interacting proteins were phosphorylated, their phosphorylated peptides were also searched for in the LC-MS/MS data used for the identification of Tor1 and Tor2-associated proteins with the Mascot program. Any phosphopeptide matched by the Mascot program was manually verified. In total, 30 phosphorylation sites were identified on Tor1, Tor2 and their associated proteins in vegetatively growing S. pombe cells (Fig. 5). This is the first comprehensive analysis of phosphorylation in the TORC, but the number of phosphorylation sites is likely underestimated because the sequences were not completely covered (Fig. 4B). Of the 30 sites, 18 were assigned to a specific amino acid residue with certainty, whereas 12 were not. The phosphorylated residues were serine or threonine.


Figure 5
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  		Figure 5  Phosphorylation sites of TOR and associating proteins. Conserved domains of indicated proteins are shown schematically as boxes and phosphorylation sites are indicated by red lines with P. Sp: S. pombe, Hs: H. sapiens. Phosphorylation sites in Hs Raptor, Hs Rictor and Hs mSin1 were taken from the high throughput phospho-proteome analysis (Olsen et al. 2006) (A) (Upper panel) Phosphorylation sites in Sp Tor2, Sp Tor1 and Hs mTOR. The domain architecture is according to Perry & Kleckner (2003), and the Pfam database. (Lower panel) Sequence comparison of S. pombe, H. sapiens and S. cerevisiae TOR kinase in the region between the catalytic domain and FATC domain. Phosphorylated tryptic peptides derived from LC-MS/MS analysis are indicated by the filled blue box. Amino acid residues with a filled red circle indicate a phosphorylation site. Amino acid residues in parentheses with open red circles or squares indicate putative phosphorylation sites that could not be assigned with certainty. Phosphorylated amino acids in mTOR are highlighted in red. (B) Phosphorylation sites in Sp Mip1 and Hs Raptor. The domain architecture is according to Kim et al. (2002) and the Pfam database. RNC: Raptor N-terminal conserved domain. (C) Phosphorylation sites in Sp Ste20 and Hs Rictor. The domain architecture is according to Sarbassov et al. 2004. (D) Phosphorylation sites in Sp Sin1 and Hs mSin1. The domain architecture is according to Schroder et al. (2007). CRIM: Conserved region in middle of all proteins in the Sin1 family, RBD: Ras binding domain, PH: Pleckstrin homology domain. (E) Phosphorylation sites of Sp Wat1, Sp Toc1, Sp Tco89 and Sp Bit61. The domain architectures of Tco89 and Bit61 are described in the supplementary figures.

 
In both Tor2 and Tor1, there were phosphorylation site(s) within the region between the catalytic domain and FATC domain at the C-terminus (Fig. 5A). The phosphorylation sites of Tor1 (S2234 or S2235) and Tor2 S2238 were similarly located just below the catalytic domain. Three phosphorylation sites of human mTOR were reported in the same region as S. pombe Tor1 and Tor2, while the sequence similarity of this region is low between fission yeast and humans (Peterson et al. 2000; Chiang & Abraham 2005; Holz & Blenis 2005). The region including the two phosphorylation sites T2446 and S2448 in human mTOR is considered as a repressor domain because the deletion of 2430–2450 mTOR results in increased mTOR activity (Sekulic et al. 2000). In addition, there was one phosphorylation site near the central FAT domain of Tor2 (Fig. 5A; Supplementary Figure S5A).

In Mip1, a homologue of human Raptor, three phosphorylation sites were clustered between HEAT repeats and WD repeats and one phosphorylation site was located in the RNC domain 1 (Fig. 5B; Supplementary Figure S5B,C). Remarkably, clustering of phosphorylation sites in human Raptor at the same region as S. pombe Mip1 was identified by high throughput phospho-proteome analysis (Olsen et al. 2006). In Ste20, a homologue of human Rictor, there were three phosphorylation sites outside of the conserved domain; two were at the N-terminus and the other one was at the C-terminus (Fig. 5C; Supplementary Figure S5E,D). Phosphorylation sites in human Rictor were also identified outside of the conserved domain (Olsen et al. 2006). Schizosaccharomyces pombe Sin1 was hyper-phosphorylated as described previously (Wilkinson et al. 1999), and there were seven phosphorylation sites in the entire protein (Fig. 5D; Supplementary Figure S5F,G). There is only one known phosphorylation site of human mSin1 at the C-terminus (Olsen et al. 2006). Identified phosphorylation sites of the remaining four components (Wat1, Toc1, Tco89 and Bit61) are schematically shown in Fig. 5E and their sequences are shown in Supplementary Figures S1 and 5H–J. There are no reports of phosphorylation sites of these homologues in other species. Although almost all phosphorylation sites identified in S. pombe Tor1, Tor2 and their interacting proteins are located in non-conserved domains among eukaryotes, the locations are quite consistent with known phosphorylation sites in humans.


   Discussion
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 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this study, we report the isolation of the S. pombe ts tor2-287 mutant that has a cellular phenotype resembling that of nitrogen starvation-induced arrested cells in complete medium at the restrictive temperature. The basic properties (loss of nutritional sensing and cell polarity, and presence of pre-replicative DNA) are similar to those of previously reported tor2 mutants (Alvarez & Moreno 2006; Uritani et al. 2006; Matsuo et al. 2007; Weisman et al. 2007). The previously reported S. pombe tor2 mutants do not have a rapamycin-hypersensitive phenotype. The tor1 deletion mutant is sensitive to rapamycin, though it remains viable (Kawai et al. 2001; Weisman & Choder 2001; Weisman et al. 2005). An additional phenotype was the failure of tor2-287 to enter the proliferative state upon nitrogen replenishment: in complete medium, cells remained small and DNA replication occurred only partially. Tor2 seems to be required for both initiating and maintaining cell growth in fission yeast.

We determined the mutation site of tor2-287 as the single amino acid substitution L2048S that locates at the near carboxy-terminus. Chromosomal integration of the L2048S mutation causes the same ts and rapamycin-sensitive phenotypes so this substitution should be the sole cause of rapamycin sensitivity. The L2048 residue resides in the catalytic domain of Tor2. We hypothesize that the mutant is inactivated at 36 °C, whereas it has an active conformation at 26 °C in the absence of rapamycin. In Fig. 6A (bottom panel), the S. pombe Tor2 sequence that contains the residue L2048 is aligned with porcine PI3 kinase {gamma} subunit, of which the 3D crystal structure is known (Walker et al. 1999). The N-lobe (red in the top panel) essential for the catalytic activity seems to be conserved between PI3K{gamma} and Tor2. The L2048 residue is located at the bottom (green circle) of the ATP-binding pocket that exists in the boundary region that connects the N- and C-lobes. The hydrophobic L2048 residue is in close proximity to the adenine base of ATP, possibly stabilizing the hydrophobic interaction with the nucleotide base.


Figure 6
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  		Figure 6  The tor2-287 mutation site in the catalytic domain and the organization of TORC1 and TORC2 complexes. (A) (Upper panel) Sequence alignment of TOR kinase and porcine PI3K{gamma} near the tor2-287 mutation site. The secondary structure of S. pombe Tor2 was predicted by Jpred (http://www.compbio.dundee.ac.uk/~www-jpred/). (Lower panel) The catalytic domain of PI3K{gamma} with bound ATP (PDB code: 1E8X) was displayed using Pymol (http://pymol.sourceforge.net/). The N-lobe and C-lobe are colored red and yellow, respectively. The ATP is indicated by the spheres. The region corresponding to L2048 in S. pombe Tor2 (885ATTIAK890 in PI3K{gamma}) is indicated in cyan. (B) Organization of the S. pombe TORC1 and TORC2 complexes. The arrows indicate the proteins that co-precipitated with Tel2, which was present in both TORC1 and TORC2. See text.

 
The substitution to hydrophilic S2048 in the mutant Tor2 protein might impair the proper interaction with ATP at 36 °C or at 26 °C in the presence of rapamycin. In the budding yeast S. cerevisiae, rapamycin sensitivity is mediated by a peptidyl-prolyl cis–trans isomerase related to human FK506-binding protein (Koltin et al. 1991). Consistently, a number of genes seem to affect cellular sensitivity to rapamycin in humans (Huang & Houghton 2001). In S. pombe, leucine uptake is regulated by Tor1, and the growth of leucine auxotroph is inhibited by rapamycin. This rapamycin sensitivity of leucine auxotrophs is suppressed by the deletion of S. pombe FKBP12 or by introducing a rapamycin-binding defective tor1 allele, suggesting that rapamycin inhibits a Tor1-dependent function and that rapamycin-FKBP12 complex is responsible for the inhibition (Weisman et al. 2005). How is the rapamycin hypersensitive phenotype of tor2-287 then interpreted in regard with the sensitivity of Tor1? Our mass spectroscopic results show that TORC1 and TORC2 share four common subunits, Tel2, Tti1 and Cka1/Orb5 in addition to Pop3/Wat1/Lst8, strongly suggesting that they are mostly likely to be functionally overlapped in many aspects of the TOR functions. Our preliminary mass spectroscopic analysis indicates that the protein level of Tor2-287 is reduced to ~15%–20% of that in wild-type at the restrictive temperature. A possible explanation of the rapamycin sensitivity of tor2-287 is that Tor1-containing TORC2 could suppress the impaired activity of Tor2-287 at the permissive temperature in the absence of rapamycin. In the presence of rapamycin, however, Tor1 (and also mutant protein Tor2-287) is inactivated so that mutant cells are unable to grow even at the permissive temperature. Alternatively, rapamycin may highly effectively inhibit Tor2-287 mutant protein, while wild-type Tor2 may be only ineffectively inhibited by rapamycin. To our knowledge, no mutation site in mammalian mTor or fungal Tor protein has been reported to cause rapamycin-hypersensitivity. Because the S. pombe Tor2 L2048S mutant site is conserved in evolution, introduction of the same or similar mutation into the PIK genes in other organisms might aid an understanding the drug sensitivity and facilitate the search for drugs targeted to Tor or PIK-related kinases.

How similar are tor2 mutant cells at the restrictive temperature to wild-type nitrogen-starved cells? The loss of Tor2 kinase activity might mimic nitrogen starvation, but the arrested tor2 mutant cells in complete medium are not identical to the nitrogen starvation-induced arrested cells. The frequency (~30%) of tor2-287 cells containing pre-replicative (1C) DNA was much lower than that (80%–90%) of wild-type nitrogen-starved cells. As the frequency of arrested cells that contain 2C post-replicative DNA reaches the maximum when cells are nitrogen-starved during mitosis-cytokinesis, the period of no increase in cell length (Mochida & Yanagida 2006), rapamycin might become highly effective during mitosis-cytokinesis in tor2-287 mutant cells.

The level of Tor2 is abundant in nitrogen-starved cells, whereas it is considered to have no role in maintaining the arrested cell state. We assume that S. pombe Tor2 is inhibited in the arrested cells, thus the identification of proteins specifically bound to Tor2 under nitrogen starvation might be of interest. Intracellular localization of GFP-Tor2 that is chromosomally integrated and expressed under the native promoter displays strong cytoplasmic signals in both vegetative and nitrogen-starved cells. In dividing cells, the cytoplasmic signals were speckled, whereas in arrested cells, the signals were both speckled and membranous. Localization of Tor2 is thus partly altered in cells from the vegetative to the nitrogen-starved cell state, though its physiologic significance is not known.

Our mass spectroscopic analysis using the FLAG-tagged Tor1 and Tor2 confirmed previous reports that Tor2 forms the TORC1 complex with Mip1/Raptor and Wat1/Lst8, whereas Tor1 independently forms the TORC2 complex with Ste20/Rictor, Sin1/Avo1 and Wat1/Lst8 (Alvarez & Moreno 2006; Matsuo et al. 2007). These proteins are indicated in the ellipsoids in Fig. 6B. In the present study, we identified six additional proteins that comprise the two classes, namely, proteins that specifically interact with either Tor1 or Tor2 and proteins that commonly bind to both Tor1 and Tor2. These specific and common proteins are shown in the rectangles in Fig. 6B. Two of the specific proteins are the presumed homologues of S. cerevisiae TCO89 and BIT61 (Reinke et al. 2004), which are associated with TORC1 and TORC2, respectively. Although the TCO89 family proteins have been found only in fungi, it is quite possible that the sequence similarity of higher eukaryotic counterparts may be below the limits of detection. We found that the BIT61 family proteins exist in human cells (LOC79899 and PRR5). Schizosaccharomyces pombe, S. cerevisiae and humans have another BIT61-like protein that was not detected in the TOR precipitates.

The remaining four proteins (three common and one specific for Tor2) are novel interacting components of the TORCs. Toc1/SPBP18G5.03 is specific to TORC1 and immunoprecipitated with Tor2 (unpublished data). It has no known sequence motif and is annotated as a sequence orphan in the database. S. japonicus has a homologue whose sequence is only weakly similar to Toc1, suggesting that Toc1 might evolutionarily change rapidly. Tel2 (Shikata et al. 2007) binds not only to Tor1 and Tor2, but also to a number of other proteins. Tel2 interacts with inositol phosphatidyl kinases, Tra1, Tra2, Tor1, Tor2, Rad3, and possibly also with Tel1. This unexpected finding suggests that Tel2 interacts with the common domain of these PIKKs. Tel2 might form a dimer with an uncharacterized protein, Tti1/SPCC622.13c, as the level of Tti1 co-precipitated with Tel2 was comparable to that of Tel2. Tel2 also interacts with Tti2/SPBC1604.17c, which is conserved in fungi and is possibly involved in mitochondrial function. The physiologic link to TORCs is unknown. Cka1/Orb5 is the casein kinase II in S. pombe, essential for viability, and the loss of Cka1 produces round shaped cells (Snell & Nurse 1994). Cka1/Orb5 might phosphorylate TORCs to maintain the cell shape. An emerging concept from the present study is that TORC1 and TORC2 complexes might be regulated in identical ways through various common subunits, although the common functions of these two TORCs are unclear in S. pombe. In addition, the TORCs, or at least their subpopulations, are larger than those considered by the previously identified protein subunits, and are under broader regulation through the common subunits.

Tor1, Tor2 and their interacting proteins so far examined in this study are all multiply phosphorylated in asynchronously growing cells, consistent with the notion that the TORC1 and TORC2 complexes are regulated in the network of protein phosphorylation and dephosphorylation. As none of the determined or candidate phosphorylation sites fits to the consensus sequence for Cdc2 kinase, the cell cycle control of the TORCs, if any, might not be under the direct control of Cdc2. Alternatively, Cdc2 phosphorylation of TOR might be detected only in mitotic extracts. Actually, Tor1 and Tor2 each have one Cdc2 site. In mammalian cells, the mTOR subunit appears to be phosphorylated by PKB and AMPK. It remains to be determined whether S. pombe TOR proteins are phosphorylated by a similar signaling mechanism.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Strains, materials and general techniques

Schizosaccharomyces pombe heterothallic haploids 972 h–, 975 h+ and their derivatives were used. Complete YPD (1% yeast extract, 2% polypeptone and 2% glucose) and minimal EMM2 (Moreno et al. 1991) were used to culture S. pombe. The nitrogen-starved cells designated G0 were prepared as follows (Su et al. 1996). The cells were grown in EMM2 to a concentration of 5 x 106 cells/mL at 26 °C. They were harvested by vacuum filtration using a nitrocellulose membrane (0.45 µm pore size; Millipore, Billerica, MA), washed in EMM2–N (EMM2 lacking a nitrogen source) once on the membrane, and then re-suspended in EMM2–N at a concentration of 5 x 106 cells/mL and incubated at 26 °C for 24 h. The G0 cells after ~2 divisions during the first 4–5 h under nitrogen starvation became small and spherical (Su et al. 1996). Nitrogen was replenished by adding fresh EMM2 medium to the concentration of 1 x 106 cells/mL. All strains used for nitrogen starvation were prototrophs. FACScan analysis was performed as described previously (Su et al. 1996). Rapamycin was obtained from Sigma-Aldrich Co., St. Louis, MO. For Western blotting, anti-TAT1 (a gift from Dr Keith Gull), anti-myc 9E10 (Calbiochem, San Diego, CA) and anti-FLAG M2 (Sigma) were used.

Construction of yeast strains

For construction of the tor2-L2048S[KanR] strain, the N-terminal truncated tor2-L2048S gene was cloned into the pFA6a-kanMX6 derivative plasmid, followed by integration into the endogenous tor2 locus. To tag the N terminus of tor2+, we first constructed the plasmid containing the kanMX6 marker flanked by the 1.0-kb upstream of the tor2 open reading frame (ORF) and 1.4 kb corresponding to the N terminus of tor2+ ORF with its own promoter. 3FLAG or GFP was inserted at the N terminus of the coding region of tor2+ in this plasmid, followed by integration into the endogenous tor2 locus. A similar strategy was used to construct the FLAG-tor1 strain using the hphMX6 marker. To tag genomic tel2+ with a sequence encoding three copies of the FLAG epitope at the C-terminus, the tel2+ ORF was amplified by PCR and cloned into pUC-3FLAG-ura4, which contains three copies of the FLAG epitope and the ura4+ marker. The resultant plasmid was linearized at the HincII site in tel2+ and used for transformation. Tel2–3FLAG function was confirmed by the cell viability.

Microscopy

DAPI staining was performed as described previously (Adachi & Yanagida 1989). Cells were fixed with 2.5% glutaraldehyde for 20 min on ice, washed 3 times with phosphate buffered saline (PBS), and observed under a fluorescence microscope after staining with DAPI (25 µg/mL). Cells expressing GFP-Tor2 were observed as described previously (Hayashi et al. 2004). Cells were fixed by immersion in 100% methanol at –80 °C. The fixed cells were washed with PBS and observed after staining with DAPI (0.5 µg/mL).

Immunopurification

Exponentially growing cells (1 x 1011) of FLAG-tor2 or FLAG-tor1 strains were lysed in the extraction buffer (25 mM HEPES–KOH pH 7.5, 200 mM NaCl, 10% glycerol, 0.1% NP-40, 1 mM phenylmethylsulfonyl fluoride) supplemented with protease inhibitor cocktail (Sigma). Extracts were centrifuged twice (20 min at 7600 rpm and 30 min at 20 000 rpm), and incubated with anti-FLAG M2 affinity gel (Sigma) for 2 h. The beads were then washed with the extraction buffer. Eluates were obtained by incubating with 150 µg/mL 3 x FLAG peptide (Sigma).

Mass spectrometry

The procedures were performed essentially as previously described (Obuse et al. 2004). Immunopurified samples were separated on a 12.5% SDS-PAGE, and visualized with Coomassie Brilliant Blue staining. The area from the top to the bottom of the separation gel was cut at ~1 to 2-mm intervals. After in-gel digestion with modified trypsin (Roche, Nutley, NJ), the resulting peptides were analyzed by online LC-MS/MS on a Finnigan LTQ (Thermo Fisher). All MSMS spectra were searched against the S. pombe non-redundant protein database including common contaminants such as trypsin and keratin with the Mascot program (Matrix Science, London, UK). The output data from Mascot was analyzed using in-house software to select reliable peptides and calculate emPAI values. emPAI values were calculated according to Ishihama et al. (2005), except that the predicted retention time was not used and the mass range for observable peptides was 400–4200. To determine proteins that specifically co-immunoprecipitated with a bait protein, the ratios of emPAI derived from bait and control proteins were used. Phosphopeptides identified by Mascot was verified manually using MSIGHT, ASCORE, and in-house software (Palagi et al. 2005; Beausoleil et al. 2006). To compare phosphorylation data from S. pombe, the information of phosphorylation sites in the human TOR components were obtained from the Swissprot database and PHOSIDA <http://www.phosida.com>.


   Acknowledgements
 
We thank Dr Miho Shikata for technical assistance. We are also grateful to Dr Keith Gull for providing the antibody and Dr Juraj Gregan for providing the plasmids. OIST was partially funded by the Japan Science and Technology Agency until the OIST Corporation started. This work was partially supported by a Grant-in-Aid for Young Scientists (Start-up) (to K. N.) from Japan Society for Promotion of Science, a Grant-in-Aid for Scientific Research on Priority Areas (to Y. N.), and a Grant-in-Aid for Scientific Research on Priority Areas (to J. K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Fumio Hanaoka

aThese authors have contributed equally to this article. Back

* Correspondence: E-mail: nagao{at}oist.jp or yanagida{at}kozo.lif.kyoto-u.ac.jp


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Accepted: 18 September 2007




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